The ability to perform parallel microanalysis on minute quantities of sample is important to the advancement of chemistry, biology, drug discovery and medicine. Today, the traditional 1536-well microtiter plate has been surpassed by microwell arrays which have an even greater number of reaction chambers and use lesser amounts of reagents due to efforts focused on maximizing time and cost efficiencies. Although there are several types of microwell arrays available, many microwell materials prove to be incompatible with the components of bioassays and chemical reactions and result in problems such as low sensitivity, high background signal, and lack of reproducibility. Thus, there continues to be a need for the development of improved microwell arrays.
One solution to the problem of incompatible materials is to apply a thin film coating of a compatible material to the microwell array to enhance its surface properties and function. Patil, et al. U.S. Pat. No. 6,395,483 has disclosed a method to coat polymeric substrates with mask layers comprised of metallic and metal-oxide for use in high-density microarray applications. Yon-Hin, et al. U.S. Pat. No. 6,440,645 has described a process to use a photoimageable thin film on a polymer substrate to form microwells or channels. Heller, et al. U.S. Pat. No. 5,632,957 describes the deposition of metal, insulator and passivation coatings of substrates to form microelectrode arrays, and to form wells over the individual microelectrodes. Walt, et al. U.S. Pat. No. 6,377,721 discloses the coating of microwell interior surfaces on fiber optic arrays with a thin film of a biologically compatible material, citing biopolymers, synthetic polymers, and metals as examples.
Certain fiber optic bundles have been used to create arrays. Several methods are known in the art for attaching functional groups (and detecting the attached functional groups) to reaction chambers etched in the ends of fiber optic bundles. See, e.g., Michael, et al., Anal. Chem. 70: 1242-1248 (1998); Ferguson, et al., Nature Biotechnology 14: 1681-1684 (1996); Healey and Walt, Anal. Chem. 69: 2213-2216 (1997). A pattern of reactive functional groups can also be created in the reaction chamber, using photolithographic techniques similar to those used in the generation of a pattern of reaction pads on a planar support. See, Healey, et al., Science 269: 1078-1080 (1995); Munkholm and Walt, Anal. Chem. 58: 1427-1430 (1986), and Bronk, et al., Anal. Chem. 67: 2750-2757 (1995).
An array of functional groups on a substrate can be constructed using lithographic techniques commonly used in the construction of electronic integrated circuits as described in, e.g., techniques for attachment described in U.S. Pat. Nos. 5,143,854, 5,445,934, 5,744,305, and 5,800,992; Chee et al., Science 274: 610-614 (1996); Fodor et al., Nature 364: 555-556 (1993); Fodor et al., Science 251: 767-773 (1991); Gushin, et al., Anal. Biochem. 250: 203-211 (1997); Kinosita et al., Cell 93: 21-24 (1998); Kato-Yamada et al., J. Biol. Chem. 273: 19375-19377 (1998); and Yasuda et al., Cell 93: 1117-1124 (1998). Photolithography and electron beam lithography sensitize the substrate with a functional group that allows attachment of a reactant (e.g., proteins or nucleic acids). See e.g., Service, Science 283: 27-28 (1999); Rai-Choudhury, HANDBOOK OF MICROLITHOGRAPHY, MICROMACHNING, AND MICROFABRICATION, VOLUME I: MICROLITHOGRAPHY, Volume PM39, SPIE Press (1997). Alternatively, an array of functional groups can be generated using thin film technology as described in Zasadzinski et al., Science 263: 1726-1733 (1994).
One major disadvantage of this type of fiber optic array is the constraint of the materials comprising the fiber optic bundle. To act as an efficient waveguide, each fiber element must consist of a high refractive index core surrounded by a low refractive index cladding. These fiber optic materials are often incompatible with many reaction conditions, particularly assays which are often conducted in aqueous solutions and contain sensitive enzymatic reagents. Two major sources of incompatibility are the dissolution of the fiber optic substrate into the solution contained in the reaction chamber and the actual chemical reaction of the fiber optic substrate with components contained in the chamber. For example, core components, such as barium and lanthanum oxides, can form hydroxides which are water soluble, particularly at elevated temperatures. Multivalent heavy metals, such as barium and lanthanum, can interact unfavorably with enzymes, especially those enzymes with metal ion co-factors. Heavy metal oxide surfaces tend to be positively charged at the solution interface, and tend to non-specifically bind negatively charged species such as nucleic acids. All of these effects will tend to degrade the performance of assays and reactions conducted in the fiber optic reaction chambers. Increasing miniaturization also tends to exacerbate these unfavorable effects.
The fact that the fiber optic substrate must be comprised of two materials (core and cladding) also can limit the effectiveness of any surface modification of the reaction chambers with a monolayer (e.g. functional groups). For example, a singly charged surface can be modified by binding to the charged surface of functionalized polyelectrolytes which contain an opposite charge. The core and cladding materials of the fiber optic substrate each have different types of charges. Thus, any modification of the fiber optic substrate with a single polyelectrolyte is impossible since the substrate does not contain a single uniform charge.
One way to alleviate these problems is to coat the array substrate with a thin film using methods described herein. In general, the discussion herein is focused on fiber optic substrates, although other substrates as described below may be used in any embodiment described herein.
Due to technical difficulties in currently used processes for coating arrays, the range of assays that can be conducted in microwell arrays remains less than would be desired. Accordingly, there is a clear need for microwell arrays which are compatible for any assay or reaction conditions.